Rickettsia rickettsii, an obligate intracellular bacterium and etiologic agent of Rocky Mountain spotted fever, infects and proliferates predominantly within vascular endothelial cells, which respond by activating a series of distinct signal transduction pathways. R. rickettsii infection of endothelial cells results in the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor which controls the expression of an array of genes involved in bacterial infections, immune response, and apoptosis. The anti-apoptotic functions of NF-kappaB are critical for the protection of host cells from apoptotic death during R. rickettsii infection. The goal of this application is to further our understanding of signaling mechanisms underlying Rickettsia-induced transcriptional activation, to evaluate their participation in the host cell response to infection, and to investigate if interfering with these signals affects rickettsial replication. Aim 1 will characterize the activation of IkappaB kinase complex (IKK) and phosphorylation/degradation of IkappaB proteins during infection. We will determine the kinetics of activation of catalytic subunits, IKKalpha and IKKbeta by an immunoprecipitation (IP): kinase assay. The role of the regulatory subunit, IKKgamma, will be evaluated using a specific, cell permeable peptide, which blocks its association with the IKK complex. The effects of selected, specific inhibitors of IKK and NF-kappaB on replication of Rickettsia organisms will also be studied. Aim 2 will investigate the activation of mitogen activated protein (MAP) kinases and their involvement in rickettsial invasion of endothelial cells and activation of NF-kappaB. Modulation of MAP kinase cascades, ERK1/2 and p38, will be examined by western blotting and immunostaining using phosphorylation state specific antibodies and activity assays by IP:western analysis. Aim 3 will define the regulation of chemokine induction in response to infection and explore its dependence on the MAP kinase and NF-kappaB pathways. Using specialized techniques of molecular biology and microscopy and species/strains of Rickettsia with varying pathogenicity, we will investigate the correlation between infection, activation of IKK/NF-kappaB and MAP kinases, and induction of chemokine response. These studies will offer important perspectives in our understanding of rickettsial pathogenesis and may lead to the identification of novel targets for supplemental chemotherapy.